The long term research goals of our laboratory center on defining the signaling events that coordinate the activity of individual endothelial cells (EC) and smooth muscle cells (SMC) along microvessels that regulate blood flow to active tissue. Our working hypothesis is that the local control of blood flow represents a coordinated activity among the cells that comprise the walls of arterioles in microvascular resistance networks. Stimulating with acetylcholine (ACh) initiates signals that propagate from cell to cell along the endothelium to initiate SMC relaxation and thereby produce vasodilation. Our laboratory has shown that ACh initiates a bi-directional Ca2+ wave that propagates at -0.1 millimeter per second over distances of several hundred micrometers and stimulates the release of nitric oxide to promote vasodilation. My preliminary experiments have revealed a novel 'Fast Calcium Response (FOR)'that travels much more rapidly (millimeters per second) and for far greater distances but only in the direction of blood flow. The project described herein is focused on understanding how this novel FOR is initiated and propagated along a vessel. Experiments are performed in vivo using anesthetized transgenic mice expressing a Ca2+ indicator protein (GCaMP2) targeted specifically to arteriolar endothelial cells. The mechanism of FOR initiation and propagation are unknown. Aim 1 will determine how the FOR is initiated by testing a variety of endothelium-dependent vasodilators (e.g. ACh, bradykinin, substance P, and ATP). I will determine whether entry of the vasodilator agonist into the blood stream and its convection along the flow path can explain the FOR. Alternatively, a secondary substance may be produced in response to such agonists that in turn triggers the FOR. I will resolve this question using microoclusion to control blood flow distribution in arteriolar networks and by microperfusing defined segments with specific antagonists. Aim 2 will determine how the FOR is actually propagated in relation to the movement of blood. By introducing fluorescent tracers of blood flow (labeled microspheres and red blood cells), I will determine the relationship between FOR and the velocity and distribution of blood flow. Resolving the nature of the FCR will provide new insight for considering how this previously unrecognized signaling pathway is regulated in vivo. In turn, this unique understanding may be applied to developing new therapeutic approaches for treating such pathophysiological conditions such as diabetes, hypertension and ischemia, where, the functional integrity of microvascular EC and SMC are compromised. My overall goal is to apply the findings of my research to understanding and treating cardiovascular disease.